The nicotinic acetylcholine receptor (AchR) will undoubtedly be the first ion channel whose architecture and function will be understood at the atomic level. Present models of this prototypic protein explain elegantly a large body of data and yet, they are incomplete andoften contradictory. Much has been learned from studies of noncompetitive inhibitors (NCIs) which bind at several interacting sites inside theion channel, at the interface of receptor protein with membrane lipids of at still undetermined sites. This project will study the high-affinity site for NCIs using a strategy based on two recent discoveries: 1. A family of uncharged, hydrophobic noncompetitive inhibitors, the cembranoids, that compete for binding with the channel blocker phencyclidine (PCP). 2. The observation that PCP displays higher affinity for the AchR from electrocyte than that from myocyte. This species difference is much smaller for the cembranoid eupalmerin acetate (EUAC). We hypothesize that PCP binds to the amino acids (a.a.) at positions 6 and 10 of the M2 transmembrane segments, and that the PCP affinity difference between receptors is due to three amino acid substitutions at those positions. We further hypothesize that EUAC binds to a.a. at M2 positions 10 and 13, which overlap with the PCP site but display little difference between the two receptors. The following experiments are proposed to test these hypotheses: I. Binding studies. Measurements of radioligand binding to AchR-rich membranes from Torpedo electric organ will be used to determine the potency rank for 20 cembranoids and the relationship of their binding site to those of inhibitors. II. Electrophysiological studies on wild-type receptors. The AchR from electric organ will be expressed in Xenopus laevis oocytes and the inhibition by cembranoids of Ach-induced currents will be studied using a voltage-clamp technique. III. Studies on modified receptors. Hybrid AchR's, constructed from subunits of muscle and electric organ receptors, will be expressed in oocytes. The affinity of these hybrids for PCP will be tested. Subsequently, individual amino acids at positions 6, 10, 13, and others of M2 segments will be modified by site-directed mutagenesis to assess the contribution of each amino acid to the binding energies of PCP and EUAC.